Abstract

Integration of ultrastructural and molecular sequence data has revealed six supergroups of eukaryote organisms (excavates, Rhizaria, chromalveolates, Plantae, Amoebozoa and opisthokonts), and the root of the eukaryote evolutionary tree is suggested to lie between unikonts (Amoebozoa, opisthokonts) and bikonts (the other supergroups). However, some smaller lineages remain of uncertain affinity. One of these unassigned taxa is the anaerobic, free-living, amoeboid flagellate Breviata anathema, which is of key significance as it is unclear whether it is a unikont (i.e. possibly the deepest branching amoebozoan) or a bikont. To establish its evolutionary position, we sequenced thousands of Breviata genes and calculated trees using 78 protein sequences. Our trees and specific substitutions in the 18S RNA sequence indicate that Breviata is related to other Amoebozoa, thereby significantly increasing the cellular diversity of this phylum and establishing Breviata as a deep-branching unikont. We discuss the implications of these results for the ancestral state of Amoebozoa and eukaryotes generally, demonstrating that phylogenomics of phylogenetically ‘nomadic’ species can elucidate key questions in eukaryote evolution. Furthermore, mitochondrial genes among the Breviata ESTs demonstrate that Breviata probably contains a modified anaerobic mitochondrion. With these findings, remnants of mitochondria have been detected in all putatively deep-branching amitochondriate organisms.

Bikonts were defined as all eukaryotes ancestrally having two centrioles and cilia, with the anterior one being the younger and undergoing ciliary transformation to become the posterior cilium with a modified structure in its second cell cycle (Cavalier-Smith 2002). Unikonts were proposed to have had a last common ancestor with only one centriole and one cilium. It has long been known that many unikonts have two centrioles and some even two cilia but these were considered derived complications. When unikonts have two cilia, the anterior one never transforms into the posterior one. As many bikonts are secondarily uniciliate, the unikont/bikont distinction stresses fundamental differences in centriolar development and inferred ancestral state, not the number of centrioles or cilia per cell, which is evolutionarily more labile. Based on a rare gene fusion and other molecular cladistic characters, as well as basic differences in microtubular cytoskeleton and ciliary development (Cavalier-Smith 2002), the root of the eukaryote tree of life was proposed to lie between bikonts and unikonts (Stechmann & Cavalier-Smith 2002, 2003; Richards & Cavalier-Smith 2005). All recent multigene trees (e.g. Burki et al. 2007; Rodríguez-Ezpeleta et al. 2007) strongly support a bipartition of eukaryotes into unikonts and bikonts and are compatible with the root lying between them, though a recent paper on just a few genes raises a potential problem for the simplest interpretation of these data (Kim et al. 2006). To test it more thoroughly and better eliminate alternatives, additional putatively derived cladistic characters need to be identified (Rodríguez-Ezpeleta et al. 2007), and other little studied lineages must be included in multigene analyses.

We focus here on the phylogenomics of one such key lineage, the breviate amoeboflagellates (Cavalier-Smith et al. 2004)—a group that has defied placement in either unikonts or bikonts or any of the six eukaryotic supergroups, and whose correct placement is likely to illuminate the primary eukaryotic divergence.

Breviata anathema (previously misidentified as Mastigamoeba invertens) is a deeply branching anaerobic amoeboflagellate eukaryote, which has been notoriously difficult to place phylogenetically (Cavalier-Smith et al. 2004; Walker et al. 2006), and has some apparent morphological affinities with unikonts (i.e. its amoeboid cell body and single flagellum) and some with bikonts (two basal bodies); its filose pseudopodia (micrographs, figure 1) differ from those of either group. In single-gene phylogenetic analyses of the small subunit ribosomal RNA gene (18S) and the largest subunit of DNA-dependent RNA polymerase II (RPB1), the position of Breviata is very unstable; it variably associates with the excavates, apusomonads (themselves either excavates or still earlier branching bikonts) and/or planomonads (formerly misidentified as Ancyromonas; see Cavalier-Smith et al. 2008) or with Amoebozoa, but no position is significantly supported (Bolivar et al. 2001; Cavalier-Smith et al. 2004; Walker et al. 2006). Amoebozoa, the group to which we now show Breviata belongs, is probably one of the earliest branches from the eukaryotic cenancestor and important for deducing its characteristics (Cavalier-Smith 2002; Richards & Cavalier-Smith 2005). Although the name Amoebozoa is old (Lühe 1913), it has only recently been recognized as a phylogenetically coherent group, with many unrelated amoebae now being excluded (Cavalier-Smith 1998; Cavalier-Smith & Chao 2003) and its classification revised (Cavalier-Smith et al. 2004; Nikolaev et al. 2006). Amoebozoa currently include classical naked and testate lobose amoebae, anaerobic Archamoebae (Entamoebae and pelobionts) and mycetozoan slime moulds (Cavalier-Smith et al. 2004), but exclude all amoeboid protozoa with true filopodia (ones that draw the cell forward by contraction), which instead belong to the bikont phylum Cercozoa that includes the chlorarachnean algae (Cavalier-Smith & Chao 2003). However, based on phylogenetic analyses and ultrastructural features, Cavalier-Smith et al. (2004) proposed a new class Breviatea including Breviata and two environmental sequences that clustered together with Breviata in 18S rRNA phylogenies, and postulated breviates as the out-group to all other Amoebozoa.

In vivo morphology of B. anathema. Light micrographs of unstained living B. anathema cells. (a) 400× DIC image highlighting the numerous branching pseudopodia and widened cell sheath at the base of the single flagellum. (b) Inset 630× DIC image showing the position of the nucleus containing a centrally located nucleolus. (c) 400× phase-contrast image highlighting the flattened pseudopodial attachments to the substrate. Scale bars, 5 μm.

As multigene analyses usually generate more robust phylogenetic inferences than single genes (Bapteste et al. 2002; Burki et al. 2007), we constructed a cDNA library from B. anathema and sequenced approximately 4100 clones and reconstructed global eukaryote phylogeny using approximately 17 300 amino acid characters (figure 2). We also searched our database for mitochondria-related genes, as Breviata is also of special evolutionary interest as an anaerobic/microaerophilic organism with unusual hydrogenosome-like organelles, whose putative mitochondrial nature is controversial (Walker et al. 2006). As is well known, several eukaryote lineages within fungi, Amoebozoa (pelobionts, Entamoeba), ciliates, heterokonts (Blastocystis) and excavates (Heterolobosea, Preaxostyla, parabasalids, diplomonads and Carpediemonas) independently modified their mitochondria into anaerobic energy-generating organelles (hydrogenosomes) or the more degenerate mitosomes (Tielens et al. 2002; van der Giezen & Tovar 2005; Barbera et al. 2007). Since all groups other than breviates that putatively represented descendants of a pre-mitochondrial eukaryotic lineage have now been investigated and shown to contain mitochondrial-related remnants (i.e. organelles or genes) (Hampl et al. 2008), the only remaining known lineage that might be ancestrally amitochondriate is the breviates.

A global phylogeny of eukaryotes. Maximum-likelihood tree with bootstrap support values (BV) from an amino acid alignment of 78 concatenated genes (17 283 characters) inferred using Raxml and Treefinder (both giving identical topology; the raxml tree is shown). Bayesian PP support values for bipartitions are also shown if more than 0.50. Filled circles denote support values of 100% BV and 1.0 PP, and dash (−) denotes support value below 50% BV or 0.50 PP. Nodes without denotation received less than 50% BV and less than 0.50 PP.

However, genes that trace their ancestry to the mitochondrion clearly demonstrate a mitochondrial history for Breviata.

2. Material and methods

(a) Library construction and EST sequencing

B. anathema (strain ATCC 50338) was cultured with one or two unidentified bacteria as food in tightly sealed 500 ml tissue culture flasks containing 75 ml ATCC 1773 medium at room temperature (approx. 21°C). Total RNA was isolated from cells harvested by centrifugation using Tri reagent (Sigma-Aldrich, St Louis, MO, USA). A non-normalized, directional, ‘microquantity cDNA library’ was constructed in the plasmid vector pAGEN-1 by Agencourt Bioscience, Corp. (Beverly, MA, USA). Approximately, 4100 randomly picked clones were 5′-end sequenced; the EST sequences were subsequently quality checked and assembled to contigs using a Phred/Phrap pipeline at the freely available Bioportal service at the University of Oslo (http://www.bioportal.uio.no).

(b) Multigene alignment construction

BLASTx analyses (http://www.ncbi.nlm.nih.gov/BLAST) of Breviata singletons and contigs were performed to identify gene similarities. Breviata sequences and significant hits (E-value >1e−5) from a range of other publicly available sequences from different databases (TBestDB, http://tbestdb.bcm.umontreal.ca/searches/login.php; NCBIest and NCBInr database) were added to the existing single-gene alignments (Rodríguez-Ezpeleta et al. 2005; Burki et al. 2007). Ambiguously aligned characters were selected manually and excluded from the analyses. For each single-gene alignment, orthologous gene copies were identified by manual inspection of phylogenetic trees and bootstrap values (BV) inferred with PhyML (rtREV substitution model, 100 bootstrap replicates; Guindon & Gascuel 2003). Additionally, for taxa with two or more nearly identical sequences, the sequence displaying the shortest branch length on the tree was kept. The final multigene dataset contained 78 genes (17 280 amino acid characters) and 37 taxa. Taxa sampled were chosen to reflect the evolutionary range of eukaryotes, and the genes selected are based on the genes detected in the Breviata library. Details about taxon sampling and genes used in the analyses are given in table S1 in the electronic supplementary material.

Three fast-evolving excavates were excluded from the main analyses owing to their long branches (Simpson et al. 2006), known to cause long-branch attraction artefacts in phylogenetic trees (Philippe 2000), but were included in an additional analysis shown in supplementary material (see figure S1 in the electronic supplementary material).

The impact of fast-evolving sites on the phylogeny was assessed by estimation with codonML in PAML (Yang 2007) under eight rate categories and subsequent site removal script applied to the alignment (S. Kumar, Å, Skjævelend, T. Ruden, A. Botnen & K. Shalchian-Tabrizi 2008, unpublished data). ML bootstrap consensus trees were inferred (as described below) from 100 pseudoreplicate datasets after the three fastest site-rate categories were removed (see figure S2 in the electronic supplementary material). Support for Amoebozoa and for the position of Breviata in optimal trees is shown in figure 3.

The placement of Breviata within Amoebozoa in three maximum-likelihood phylogenies with BV inferred with Raxml after removing categories of fast-evolving sites. Only the Amoebozoa branch is shown and global trees are shown in figure S2 in the electronic supplementary material. Categories 6, 7 and 8 refer to the sites removed; category 8 comprises the fastest evolving sites. Filled circles denote support values of 100% BV.

(c) Phylogenetic analyses and approximately unbiased test

All phylogenetic analyses were performed on the Bioportal at the University of Oslo (http://www.bioportal.uio.no). Maximum-likelihood phylogeny of the concatenated data was inferred with raxml MPI v. 2.2.3 (Stamatakis 2006) and Treefinder (Jobb et al. 2004) The rtREV+F evolutionary model was preferred by ProtTest v. 1.3 under the Akaike information criterion with four GAMMA rate categories (Posada & Crandall 1998). Topological tree searches were performed with 100 randomly generated starting trees, while bootstrap analysis was performed on 100 pseudoreplicates and one random starting tree for each replicate, with the same evolutionary model as the initial search. In the raxml, analyses trees were inferred under Protmix (Stamatakis 2006).

Bayesian inference used PhyloBayes v. 2.3 (Lartillot & Philippe 2004), with the CAT evolutionary model, a gamma-distributed across-site variation (four discrete rate categories) and random starting tree. Changes in log likelihood as a function of time were used to estimate whether the two parallel chains had reached a stationary state. This was then used to set the burn-in and compare the frequency of the bipartitions between several independent runs. The largest discrepancy (maxdiff) between the bipartitions was less than 0.1, and therefore we considered the Markov chain Monte Carlo chains to have converged. The tree and PP values presented in figure 2 are a consensus of the cold chains from the two independent runs.

The approximately unbiased (AU) tests were performed on the dataset that included all sites and on datasets with categories of fast-evolving sites removed (see table S2 in the electronic supplementary material). Site likelihoods were calculated in Raxml and the AU test performed with CONSEL (Shimodaira & Hasegawa 2001) using the rtREV evolutionary model, default scaling and replicate values.

3. Results and discussion

(a) A global phylogeny including B. anathema

In our phylogeny, Breviata is convincingly placed with Amoebozoa (supported with 87/88% BV and 0.97 PP value; figure 2) by both maximum-likelihood (inferred with Raxml and Treefinder, respectively) and Bayesian methods. Removing the fastest evolving sites of the alignment did not influence this placement (figure 3; see figure S2 in the electronic supplementary material). Removing the fastest evolving sites increased the bootstrap support to 100 per cent BV for Breviata grouping with Amoebozoa (figure 3a). Sequential removal of additional fast-site categories decreased the support for most supergroups, including Amoebozoa, but the relationship of Breviata+Amoebozoa was always recovered. In all trees with fastest evolving sites removed, the clear-cut separation into unikonts and bikonts (with Breviata among the unikonts) was even more strongly supported than that shown in figure 2 (88, 97, 95% BV; see figure S2 in the electronic supplementary material). An additional phylogeny including three additional fast-evolving excavate taxa (Giardia intestinalis, Trichomonas vaginalis and Trimastix pyriformis; see figure S1 in the electronic supplementary material) also supported the placement of Breviata with Amoebozoa, but somewhat less strongly. Hence, this relationship is robust and not sensitive to the removal of fast-evolving sites or to taxon sampling. The alternative placement of Breviata within bikonts suggested by many single-gene trees (Cavalier-Smith et al. 2004; Shalchian-Tabrizi et al. 2006; Walker et al. 2006) is not seen in any inferred multigene trees, and this topology was rejected by the AU tests of the reduced datasets from which the fastest evolving sites were successively removed (AU test; see table S2 in the electronic supplementary material).

Although grouping of Breviata with Amoebozoa is strong, bootstrap support for placing Breviata as a sister to—rather than among—the other amoebozoan taxa is weak. Accordingly, the AU tests did not reject the possibility that Breviata may branch among other Amoebozoa as sister to the other anaerobic amoebae (Archamoebae: Entamoeba and Mastigamoeba; see table S2 in the electronic supplementary material) and this sister relationship is supported in two of the trees inferred after removing fast-evolving sites (figure 3a,c). However, it is more likely that Breviata is sister to the other Amoebozoa, owing to its lack of four sequence signatures in the 18S rRNA gene that other Amoebozoa all share; single nucleotide substitutions at positions 385, 777 and 1010 and a 1–2 nucleotide insertion in the loop between positions 1060 and 1064 (Fahrni et al. 2003). If Breviatea were sisters to Archamoebae, all four signatures must have reverted to the ancestral state found in all out-groups to Amoebozoa (Fahrni et al. 2003), which is unlikely as most other Amoebozoa have all four of these signatures, and all have at least two (Fahrni et al. 2003).

A minority of 18S rRNA analyses have suggested a specific affiliation of Breviata to apusomonads (Walker et al. 2006), but too few protein-coding genes are available from apusomonads for us to test this hypothesis directly. Likewise, the phylogenetic position of apusomonads is controversial, with ultrastructural and gene fusion evidence suggesting a bikont affinity (Karpov & Zhukov 1986; Stechmann & Cavalier-Smith 2002) while two- to six-gene phylogenies place Apusomonas proboscidea as sister to opisthokonts (Kim et al. 2006). However, when α-tubulin was excluded from the multigene analyses of Kim et al. (2006), the placement of A. proboscidea as sister to Amoebozoa could not be rejected (Kim et al. 2006). Thus, there is no evidence suggesting that Breviata is misplaced in our tree.

(b) Breviate amoebae are unusual amoebozoans

In all our multigene trees, Breviata is placed with Amoebozoa with high support. The precise placement within the group, however, is not consistent in the trees inferred, as some of them support a sister relationship between Archamoebae and Breviata, while others indicate that Breviata is sister to the remaining Amoebozoa (figures 2 and 3). Notably, the absence of the Amoebozoa-specific substitutions in the 18S sequence indicates that the latter hypothesis, consistent with the hypothesis proposed by Cavalier-Smith et al. (2004), is more likely. Walker et al. (2006) reasonably argued that because Breviata is not closely similar in morphology to any of the other classes of ciliated Amoebozoa it does not belong in any of them (Walker et al. 2006). However, their conclusion that it is therefore not an amoebozoan did not take into account the possibility of a common ancestry plus later substantial morphological divergence from the other classes, which now appears to be the case. Indeed, amoebozoan morphological diversity has been expanded by careful observations that reveal a unique gait in Breviata locomotion. These amoebae travel by ‘walking’ with thin but robust leg-like pseudopodia that emanate from the anterior of the cell body, and adhere to the substratum, while the cell body proceeds forward just as a package travelling on a roller conveyor or ‘tractor on treads’ (figure 1). The filose ‘legs’ often remain as trailing filaments before they retract into the cell body. This character distinguishes Breviata from other organisms, as no other eukaryote has even vaguely similar motor movements.

Prior to the addition of Breviatea, Amoebozoa comprised two well-defined subphyla: the often ciliated Conosa (Mycetozoa, Archamoebae) characterized by a conical microtubular skeleton diverging from the centriole or centrosome, and the purely amoeboid Lobosa that lack cilia, centrioles and cytoplasmic microtubules (Cavalier-Smith 1998). Our demonstration that Breviata is an amoebozoan significantly increases the cellular diversity of the phylum owing to its unusual pseudopodial morphology, mode of locomotion and rather complex cytoskeleton. In marked contrast to the also anaerobic Archamoebae, Breviata has two centrioles and a substantially more asymmetric microtubular cytoskeleton. These differences, plus the presence of Golgi stacks in Breviata, but not Archamoebae, justify their being in separate classes (Cavalier-Smith et al. 2004; as do the four contrasting rRNA signatures mentioned above), but (contrary to Walker et al. 2006) are not enough to merit separate phyla. Thus, there are now three broadly different cytoskeletal patterns in Amoebozoa.

(c) Implications for ultrastructural evolution in early eukaryotes

The ancestral cellular structure for Amoebozoa was argued to be a uniciliate, unicentriolar amoeba with a radially symmetric pericentriolar microtubular cone (Cavalier-Smith et al. 2004). However, as the uniciliate Breviata possesses two centrioles, one of which serves as the basal body of the cilium resulting in an asymmetric cytoskeleton (Walker et al. 2006), this interpretation needs some re-evaluation. As there are also other amoebozoan lineages with two basal bodies, such as myxogastrids and a few protostelids, the two basal bodies in Breviata do not contradict the inference that Breviata is an amoebozoan, but merely suggest that it is not an Archamoeba (Cavalier-Smith et al. 2004). If Breviata were sister to Archamoebae, as some trees excluding faster evolving sites suggest but which the rRNA signatures render unlikely, one could argue more strongly that its having a second barren centriole is a derived state. However, our more inclusive trees and 18S rRNA signatures in combination indicate that Breviata is probably sister to all previously accepted Amoebozoa. This makes it harder to infer the ancestral state of Amoebozoa, in which there are now two groups with two centrioles/basal bodies (Breviata, myxogastrids), three with one centriole per kinetid (Multicilia, Phalansterium, Archamoebae) and one with a mixture (protostelids). Thus, a double centriolar ancestral state for Amoebozoa is almost as parsimonious as the single centriolar scenario (Cavalier-Smith et al. 2004), especially as deeply branching opisthokonts (chytrids and choanoflagellates), the sister group to Amoebozoa, have two centrioles. With respect to the cytoskeleton, the marked asymmetry found in B. anathema contrasts with the hypothesized symmetrical ancestral state of Amoebozoa (Cavalier-Smith 2002). This asymmetry could be secondarily derived in B. anathema and does not imply an affinity to the asymmetric bikonts since the detailed arrangement of their ciliary roots differ substantially. Thus, the inclusion of Breviata within Amoebozoa as its most divergent group has important implications for the ultrastructural evolution and likely ancestral state of the cytoskeleton and centrioles in Amoebozoa and eukaryotes generally. Our findings make it important to study both the cytoskeleton and the pattern of ciliary and centriolar development more thoroughly in B. anathema and test their generality among different breviates. As contrasting modes of ciliary development were a key aspect of the original recognition of the primary dichotomy between bikont and unikont eukaryotes (Cavalier-Smith 2002), such studies are of key significance for clarifying the basic organization of the earliest eukaryote cells. Unfortunately, ciliary development is unstudied for Breviata and for apusomonads, whose putative inclusion within unikonts (Kim et al. 2006), is unexpected, given their biciliate (not necessarily bikont; for the distinction see Cavalier-Smith 2002) nature and the structure of their ciliary roots (Molina & Nerad 1991), and needs further confirmation by multigene analyses.

(d) The mitochondria-like organelle in B. anathema was probably derived independently from the other anaerobic lineages

In our Breviata cDNA library, we identified key mitochondria-derived nuclear-encoded genes often seen in amitochondrial taxa that trace their ancestry back to an α-proteobacterial ancestor (here shown by cpn60 (see figure S3 in the electronic supplementary material) and tim17 (data not shown)). This clearly rejects the possibility that Breviata is a pre-mitochondrial eukaryote, and suggests that the dense organelles bounded by two membranes seen proximal to the nucleus in Breviata are mitochondria-related organelles. Further investigations of mitochondrial function in Breviata, including a search for hydrogenase and biochemical studies, are now needed. If Breviata is sister to other amoebozoa, the anaerobic adaption of the mitochondria in Breviata occurred independently of other known cases. However, our multigene trees and AU tests do not exclude the possibility that Archamoebae and Breviata form a single secondarily anaerobic amoebozoan clade.

All extant eukaryotes examined in detail, even anaerobic ‘amitochondriate’ eukaryotes, have nuclear genes whose phylogenetic history is best explained by entry into the eukaryote lineage with the mitochondrion endosymbiont. It is thus unlikely that the anaerobic nature of Breviata represents the ancestral state of Amoebozoa, even though our data suggest that Breviata may be the deepest diverging amoebozoan lineage. The ancestral amoebozoan must have been at least facultatively aerobic, though it could have been a facultative aerobe/anaerobe, as many have postulated for the ancestral eukaryote (Cavalier-Smith 2006). Possibly aerobic members of the Breviata clade will be discovered.

The challenging task of resolving eukaryotic global phylogeny has progressed through phylogenomic analysis of major lineages (e.g. Nikolaev et al. 2004; Rodríguez-Ezpeleta et al. 2005; Burki & Pawlowski 2006; Burki et al. 2007; Patron et al. 2007; Rodríguez-Ezpeleta et al. 2007). Here, we demonstrated that investigating single, deeply diverging nomadic species is also crucial for improving our understanding of early evolutionary history of major lineages of eukaryotes. Placing the previously unaffiliated breviates, with their unique cytoskeletal pattern, in a clade with other Amoebozoa illuminates the evolutionary diversity of Amoebozoa and raises new questions concerning the nature of ancestral amoebozoan and of the unikont–bikont bifurcation suspected to reside at the base of the eukaryote tree.

Acknowledgements

We thank Cédric Berney for helpful comments on the manuscript, Dag Klaveness for fruitful discussions and Surendra Kumar for the site removal script. The Norwegian Research Council has granted scholarships to K.S.-T., M.A.M., R.J.S.O. and research project to K.S.J. T.C.-S. thanks NERC and the Canadian Institute for Advanced Research Evolutionary Biology Program for fellowship support and NERC for research grants. The pan-Canadian collaboration Protist EST Program (PEP: http://megasun.bch.umontreal.ca/pepdb/pep.html) generated sequence data for some of the species included in the phylogenetic analyses.